ABSTRACT: the location of the CLNs, they

ABSTRACT: A hallmark feature of follicular dendritic cells (FDCs)is their ability to retain antigens and virions for a prolonged duration. FDCsin the cervical lymph nodes (CLNs) are particularly relevant in elucidatingHuman Immunodeficiency Virus (HIV) – 1 infection of the central nervous system(CNS), which are the cerebrospinal fluid (CSF) draining lymph nodes (LNs). Wepostulate that any potential HIV egress from the CNS is likely reflected in theCLN viral archive.

The CLN FDC viral reservoir like the other HIV reservoirswould contribute to both low-level viremia and viral resurgence upon cessationor failure of combined antiretroviral therapy (cART). Besides prolonged virionretention on CLN FDCs, the suboptimal penetration of cART similar to other LNsis another factor contributing to establishing this viral reservoir. Unlike theperipheral LNs and FDCs within them, the CLN FDCs have not been extensivelyinvestigated and only recently garnered attention. This interest in CLN FDCshas been driven by detailed elucidation of the meningeal lymphatic system. Asthe CSF drains through the meningeal lymphatics and nasal lymphatics via thecribriform plate, CLN FDCs may acquire HIV after capturing them from T cells,antigen presenting cells or virions in the CSF.

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In this review, we outline theunderlying mechanisms of viral accumulation on CLN FDCs and the potentialimpact on viral resurgence or achieving a cure for HIV infection.   INTRODUCTIONThe cervical lymphnodes (CLNs) are a group of lymph nodes (LNs) in the neck region that arelocated adjacent to the cervical region of the spinal cord and in closeproximity to the sternocleidomastoid muscle. Depending on the location of theCLNs, they may be classified as, a) Superficial anterior CLNs; b) Superficial posteriorCLNs; c) Superior deep CLNs; or d) Inferior deep CLNs. The glymphatics andmeningeal lymphatic system connects the central nervous system with the CLNs 1; 2; 3; 4; 5; 6; 7; 8.

Moreimportantly, T cells and antigen presenting cells (APCs) migrate with thecerebrospinal fluid (CSF) as it drains along the nasal lymphatic path throughthe cribriform plate and eventually access the CLNs (Figure 1A) 1. There is a networkof stromal cells within LNs and follicular dendritic cells (FDCs) are one of thosestromal cell types. FDCs were first identified as “antigen retaining reticularcells” 9. Subsequently, FDCs have beenrecognized for their unique ability to retain antigens for a prolonged duration10. This property of FDCs is criticalfor several immune functions, including germinal center (GC) formation andlong-term immune memory. FDCs develop from perivascular precursors of stromalcell origin, which are seeded throughout the body.

Their maturation requireslymphotoxin and tumor necrosis factor (TNF) signaling via B cells 11. FDCs are found within the B-cellfollicles (BCFs) and GCs develop in the BCFs during T cell-dependent antibodyresponse 12. As the BCFs mature into GCs, FDCsmigrate into the light zone (Figure 1B).Antigenacquisition, processing and retention by FDCs impact immune response.

FDCs retainantigens from months to years 13. However, thereis inadequate experimental data demonstrating prolonged antigen retention byFDCs. In fact, most predictions are extrapolations based on decay rates. Inaddition to prolonged antigen retention, FDCs can also similarly retain HumanImmunodeficiency Virus (HIV) – 1 (Figure1C) 14.

The FDC microenvironment is highlyconducive for HIV infection 15. Some studies suggest combinedantiretroviral therapy (cART) can clear FDC-associated virus 16. While otherstudies imply that cART does not diminish FDC HIV reservoir 17. Nonetheless, FDCsare considered a lymphoid tissue viral reservoir responsible for low-levelviremia 18 and a source of resurgent virus uponcessation of cART 19. Of note, FDCretained HIV represents a divergent viral archive 20. The CLN FDCslike the FDCs in the peripheral LNs are also a HIV/SIV reservoir. We postulatethat the FDC viral reservoir in the CSF draining CLNs represents HIV egressingfrom the CNS.

  In this review, we discusshow CLNs acquire, accumulate and transmit HIV. In addition, we present somerecent advances in FDC-related HIV research (Table 1).  CNS and CLN FDCs are importantcomponents of HIV neuroimmunopathogenesisHIV neuroinvasionoccurs very early during infection 21; 22with transmigrating infected monocytes/macrophages 23. In SIV/SHIV macaquemodels, HIV neuroinvasion occurs within a few days to weeks 24.

HIV cannot beeliminated from CNS as infected monocytes or microglia have a long lifespan andlow turnover 25. The monocytes andmicroglia support latent HIV infection 26; 27; 28; 29, therebyestablishing a viral reservoir with suboptimal penetration of cART 30; 31 across the bloodbrain barrier (BBB). Besides the CNS,HIV persists in the LNs, spleen, gut-associated lymphoid tissue (GALT),reproductive organs and lungs 32; 33. LNs are a knownreservoir of persistent HIV/SIV viral infection under suppressive cART 34; 35; 36; 37; 38; 39. Several uniquecharacteristics of the LNs contribute to HIV’s ability to persist in thistissue. For example, LN tissue has a slower decay rate than in the peripheralblood 36.

Additionally,the LN follicles contain FDCs that capture HIV virions on their cell surface inimmune complexes 40. FDCs in the peripheral LNs havebeen characterized as another viral reservoir site 18; 41; 42. Of note, HIVsusceptible TFH cells are located in close proximity to FDCs, which within peripheral LNs have been shown totrap virions in their native non-degraded state for months to years 43; 44; 45; 46 with a half-life ofapproximately 2-3 months 47; 48.

While FDC-trappedvirus does not replicate or evolve; however, it can infect nearby traffickingcells 40; 48. Even during cART, replicating viruspersists and replenishes trapped stores of HIV 19; 49. Until thedescription of glymphatics and functional meningeal lymphatic system, CNS wasconsidered to be immune-privileged 3. With the elucidation of structuraland functional features of this CNS associated lymphatic system 1; 4; 50, it is now well acceptedthat CNS undergoes constant immune surveillance in the meningeal compartment.

The meningeal lymphatic system, along with glymphatics presents a uniqueconnection between the CNS and CLNs. HIV may pass with CSF as virions, infectedT cells or APCs through the cribriform plate along the nasal lymphatic pathwayand access the CLNs. Lymph entering the CLNs through the afferent lymphatics ischanneled through the subscapular sinus (SCS) into the medulla. Thefibroblastic reticular cells (FRCs) conduits access afferent lymph and traverseBCFs, where they intersect FDCs. FRC conduits continue into the cortex wherethey terminate at high endothelial vessels or the medulla 51.Recent focus onelucidating the meningeal lymphatic system has tremendously enhanced ourunderstanding of immune surveillance in the CNS 1; 2; 3; 4; 5; 6; 7; 50.

Lymphatic vessels were firstidentified in the dura mater of rats 5. In some studies of themeningeal lymphatics 1, the system has beendescribed as part of the CNS, while others have opposing conclusions 7. This is not surprising since lymphatic vessels are component of thesurrounding connective tissue that are included in the CNS. However, lymphaticvessels can absorb CSF from adjacent subarachnoid space and brain interstitialfluid via the glymphatics. Further detailed investigations are required to fullyunderstand the functionality of CSF drainage and how it might impact HIVaccumulation within CLNs.

 Circulatingconventional DCs (cDCs) are known to traffic into the CNS in response toneuroinflammation 52; 53; 54; 55; 56; 57; 58; 59 during HIV/SIVinfection 60. WithinCNS,cDCs act as both “carrier and bearer”of HIV and contribute both to neuropathology as well as CNS reservoir. Recentstudies suggest that cDCs may capture HIV within the CNS and deliver it todifferent compartments of CLNs including FDCs 43; 44; 61; 62. The CLN FDCswould create a viral repository where virus can remain bound for prolongedduration 46.

The immune cell retrogradetransport studies 1; 4; 63; 64 provide cluesthat cDCs upon encountering HIV virions within brain would migrate along themeningeal lymphatic vessels to draining LNs (CLNs, near the brain stem) viaglymphatics delivering HIV particles to different compartments of CLNsincluding FDCs as shown for peripheral LNs 43; 44; 61. It is importantto note that CLNs are the major site for systemic activation of CNS-specific Tcells. They receive input from the CNS in the form of antigens and cDCs 65. Within CLNs, HIVviral particles could be transmitted to CD4+ T cells or trapped in FDCs, whichstabilize and protect HIV thus providing a long-term reservoir of infectiousHIV 18; 34; 36; 66. In addition,FDCs activate CD4+ T cells within GCs and increase virus production in thesecells even in the presence of cART 42; 67; 68; 69; 70. Given the recentdetailed description of the functional meningeal lymphatic system 1, it is bothtimely and significant to assess the involvement of deep CLNs in HIV neuropathogenesis.Of note, additional mechanistic studies are required to determine if the CLNFDC reservoir is an archive of CNS egressing virus.

 Key Cellular Players in the CLNCLNs like other LNs play a central role in thedevelopment of adaptive immunity against pathogens and particularly thegeneration of antigen-specific B cell responses in specialized areas withinGCs. LN pathology was recognized as an important consequence of HIV infectionsince the beginning of the epidemic. Investigation into the structural andfunctional alterations induced by HIV and SIV has further cemented the centralrole that lymphoid tissue plays in HIV/SIV pathogenesis.

The coexistence ofconstant local inflammation, altered tissue architecture, and relativeexclusion of virus-specific CD8 T cells from GCs create a unique environmentfor the virus evolution and establishment of viral reservoir in specific GCcells, namely T-follicular helper CD4 T cells (TFH). A betterunderstanding of the biology of immune cells in HIV-infected LNs is aprerequisite to attaining complete viral eradication 71.FDCs are a subsetof DCs that are essential for GC formation and production of various types ofantibodies 72. They reside insecondary lymphoid tissues such as spleen, tonsils, LNs, and follicles thatappear at extra-nodal sites 73. GCs of secondary lymphoid tissuesare composed of activated B cells, TFH cells and FDCs.

In the GCmicroenvironment, activated B cells communicate with FDCs by interacting withan antigen on their surface and then present this antigen to TFHcells. FDCs can select for B cells to re-enter the GC or exit with the help ofTFH cells 13. FDCs have aunique ability to retain immune complexes on their dendritic processes.

Theseimmune complexes consist of antigen-antibody complexes 74, which can retain infectious virionsfor several months even in the presence of neutralizing antibodies or undercART 34. FDCs interactwith TFH cells in GCs and these cells serve as a reservoir ofinfectious virus. Surrounding GC T cells become highly susceptible to infectionwith HIV X4 isolates 68. HIV productionincreases to two-fold when viral particles are transferred from FDCs tosusceptible CD4+ T cells 42. FDCs can secrete inflammatorycytokines 75 including tumornecrosis factor alpha (TNF-a) and thereby contribute to enhanced transcriptionin the LNs 42. Data has been generated tounderstand the interactions of FDCs with their GC counterparts. However, underlyingmechanisms behind FDC reservoirs remain unclear and require further studies. Severalstudies in model systems indicate that the HIV virions on FDCs remaininfectious 37.

Specifically,Fletcher et al. demonstrated that HIV virions in immune complexes on murineFDCs remain infectious ex vivo for upto nine months after being captured by FDCs 34. Thischaracteristic of the FDCs is particularly interesting because most of theidentified reservoirs of persistent virus are found in the integrated pro-viralstage of the HIV replication cycle. It is important to note that currentapproaches to eliminate persistent HIV have largely focused on elimination ofHIV pro-viral DNA. While the HIV/SIV lymphoid reservoir has beenwell-characterized utilizing RNAscope and DNAscope methodologies 76, however the CLNFDC HIV reservoir has only recently been characterized 77 and warrants further studies tofully define. TFH are a subset of CD4 Tlymphocytes. TFH play a key role in B-cell differentiation.

  TFH cells assist B cells in theproduction of antigen-specific antibodies. They are essential for memory B cellactivation, survival, and differentiation. Restriction of productive SIVreplication to these TFH cells in elite controller macaques and nottypical progressors has been demonstrated 78.During HIV infection, cellular interactionsbetween FDCs, GC B-cells and TFH cells result in reservoirestablishment. TFH associated replication competent virus may be thesource of resurgent HIV after cART interruption or failure.

As such, TFHare increasingly recognized as another major CLN associated reservoir of HIVinfection 79; 80.However, mechanisms by which these cells get infected remain unclear. TFHexpress very little CCR5 and in macaque studies it has been shown that TFHlacking CCR5 cells can be infected invivo with CCR5-tropic SIV 81; 82.In both humans and macaques, TFH infection is almost certainly resultingfrom infection of CCR5-expressing pre-TFH population 83.

Accelerating the TFH responses early during HIV/SIV infection is apromising immunotherapeutic approach. However, because TFH cells areinfected during the HIV/SIV infection and represent a reservoir, this mayinterfere with HIV vaccine strategy. Thus, TFH represent both goodand bad guys during HIV/SIV infection 84.In cART-naïve as well as treatedindividuals, TFH and GC B cells are elevated. In addition, there isa direct correlation with the activated T-cell population in LNs 85.In absence of cART, during chronic HIV infection, viral replication isconcentrated in secondary lymphoid follicles (SLF).

TFH cells havebeen shown to be highly permissive to HIV within SLF. They are the source ofreplication competent HIV during latency 86. HIV vaccines are not strong inducers of neutralizing antibodies.

However, in a study of rhesus macaques immunized with HIV envelope glycoproteintrimer there was a substantial production of HIV neutralizing antibodies 87. The high antibody titers had a strong correlation to GC B cells and TFH  87. These observations highlight the need to study more details of LNs,since in the past HIV reservoir studies have frequently focused on peripheralblood.             Anothersubset of T cells in SLF, follicular regulatory T (TFR) cells have beendescribed 88.These cells share some phenotypic characteristics with TFH cells. Ofnote, studies that demonstrated TFH permissiveness to HIV infectionalso included TFR 89.TFR express greater levels of CCR5 and CD4. They also support higher frequencyof viral replication.

Expression of Ki67, a marker of proliferative capacityappears to correlate with viral replication in these cells. As such, TFR differfrom TFH in their susceptibility to R5 HIV infection 89. Furthermore, recently, Natural killer (NK) cells have alsobeen shown to migrate into the LNs. The role of NK cells in LNs is not clear.However, studies in African green monkeys demonstrated that these cells enterand persist within the LNs. Their depletion with IL-15, resulted in an increasein viral replication thereby suggesting a key role for NK cells to establishand maintain this reservoir 90. Eradication of FDC viral reservoirA significant challenge to HIVeradication is the elimination of viral reservoirs in GC TFH cells.

GCsare considered to be immune privileged for antiviral CD8+ T cells, however unselectedCD8+ T cells engineered to express CXCR5 directs them to viral sanctuaries 91.CXCR5 expressing cytotoxic T cells are able to selectively enter BCFs anderadicate infected TFH and B cells 92.Certain population of SIV-specific CD8+ T cells expresses CXCR5 (C-X-Cchemokine receptor type 5, a chemokine receptor required for homing to GCs) andexpands in LNs following pathogenic SIV infection in a cohort of vaccinatedmacaques 93.

Animals that exhibited greater control of SIV replication had a greaterexpansion of these cells. The increase in CXCR5+ CD8 T cells was associatedwith the presence of higher frequencies of SIV-specific CD8 T cells in the GC.Following TCR-driven stimulation in vitro,CXCR5+ but not CXCR5- CD8+ T cells generated both CXCR5+ as well as CXCR5-cells. However, the addition of TGF-beta to CXCR5- CD8+ T cells induced apopulation of CXCR5+ CD8+ T cells, suggesting that this cytokine may beimportant in modulating these CXCR5+ CD8+ T cells in vivo.

Thus, CXCR5+ CD8 T cells represent a unique subset ofantiviral CD8+ T cells that expand in LNs during chronic SIV infection and mayplay a significant role in the control of pathogenic SIV infection 93.An important milestone in purging FDCreservoir was demonstrated by utilization of soluble complement receptor 2 orCD21 94.CD21 is necessary for HIV interaction with FDCs and B-cells 95.In fact, interaction of HIV with FDC stabilizes the virus 66. Despite the stabilized interaction, Heesters and coworkers were ableto purge the FDCs of HIV virions by utilizing a soluble form of CD21 94.Thus, intersecting CD21:C3d interactions significantly reduced recycling ofvirions through the endosomal compartment. In addition, viral transmission to TFHwas diminished in in vitro studies 94.

An alternate approach to purge FDC HIV reservoir involved monoclonal antibodiestargeting CD21 96.Thus, blocking CD21 interactions might purge FDC HIV reservoir.  Future PerspectivesProfound anddurable suppression of HIV by cART represents a major accomplishment inHIV/AIDS research 97; 98. However, HIVpersists in patients despite long-term administration of cART 35. Withdrawing cARTinvariably results in viral rebound 99; 100. One of themajor challenges with cART is to prevent virological failure. Studies in rhesusmacaque have demonstrated sustained virologic control with an antibody thattargets the gut-homing integrin a4b7 in combination with cART 101.

While,mechanisms underlying observed virologic suppression remain to be elucidated. Progressbeyond a functional cure has been limited to some isolated instances such asthe case of a Berlin patient 102 orexperimental strategies involving CRISPR/Cas9 mediated excision of HIV 103; 104. Whetherthese innovative strategies purge CNS and LN HIV reservoirs remain to be tested.

LifetimecART is associated with toxicity, residual chronic inflammation and theaccelerated onset of co-morbidities associated with aging. As such, betterstrategies are needed to eliminate these complications such as immunotherapy incombination with cART or any therapies, which include enhancement of innateimmunity.